Dilute surfactant or isolated surfactant protein solution for the reduction of surface tension in the lung

ABSTRACT

In permeability lung edema, cardiogenic lung edema or neonatal respiratory distress, there is heterogeneous liquid distribution throughout the lungs. The excess alveolar liquid reduces gas exchange. Mechanical ventilation is used to improve gas exchange. In the presence of heterogeneous liquid distribution, there are surface tension-dependent stress concentrations in septa separating aerated from flooded alveoli. Mechanical ventilation, by inflating the lung above normal volumes, thus increasing surface tension above normal, exacerbates the stress concentrations and consequently injures, or exacerbates pre-existing injury of, the alveolar-capillary barrier. Any means of lowering surface tension should lessen ventilation injury of the lung. In the present invention, dilute exogenous surfactant solution or surfactant protein C solution interacts with albumin to lower surface tension, likely through effective promotion of surfactant lipid adsorption. Dilute surfactant or SP-C solution could be administered via either the trachea or the vasculature. Either solution could be delivered in the absence or presence of albumin or alternative facilitating solute, to lower surface tension and lessen ventilation injury of the heterogeneously flooded lung.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/194,096 filed Jun. 27, 2016, which claims the benefit of U.S. Provisional Application No. 62/185,967 filed Jun. 29, 2015, the entire disclosures of each of the aforesaid applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The present invention was supported in part by funds from the U.S. government (NIH Grant No. RO1 HL113577), and the U.S. Government may therefore have certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 101995_043102_SeqListing.txt. The text file is 1 KB. It was created on Aug. 15, 2019 and is being submitted via EFS-Web with the filing of the specification.

FIELD OF THE INVENTION

The present invention relates to methods for minimizing mechanical ventilation injury to an edematous lung being subjected to mechanical ventilation. More particularly, the methods of the present invention comprise lowering the surface tension of liquid in alveoli of the edematous lung by providing to the alveoli a surfactant-associated protein and a negatively charged solute.

BACKGROUND OF THE INVENTION

Lung physiology. The main passageways for air to travel from the nose or mouth to the lungs are the bronchi, which eventually branch to the bronchioles, which then branch to alveolar ducts. The terminal airspaces of the lungs, the alveoli, where gas exchange takes place, branch off of the alveolar ducts. Air pressure is equal between two adjacent alveoli. Thus, as equal pressures are applied to each side of any wall, or septum, between adjacent air-filled alveoli, such septa are substantially planar in shape. The surface of the alveolus is lined with type I and II alveolar epithelial cells, on top of which there is a thin liquid lining layer. Thus, there is an air-liquid interface in the lungs that has an associated surface tension. Alveolar type II epithelial cells release surfactant, which adsorbs to the interface and maintains low surface tension in the lungs. Lung surfactant is a mixture of phospholipids, the most abundant of which is dipalmitoylphosphatidylcholine (DPPC); neutral lipids; and four surfactant-associated proteins, surfactant protein (SP)-A, SP-B, SP-C and SP-D. Surfactant proteins B and C, which are hydrophobic, facilitate surfactant lipid adsorption. By lowering surface tension, surfactant reduces the pressure required to keep the lungs inflated and reduces the work of breathing.

Inside of alveolar septa are located the pulmonary capillaries. The tissue and liquid between capillary blood and alveolar air constitute the alveolar capillary barrier, across which gas exchange occurs.

Acute respiratory distress syndrome (ARDS) and ventilation-induced lung injury. The acute respiratory distress syndrome can be caused by any number of different initial insults. Regardless of cause, with ARDS there is inflammation in the lungs. With inflammation, there is increased permeability of the alveolar-capillary barrier and liquid leaks out of the blood vessels. The liquid carries with it plasma proteins—principally the most abundant plasma protein, albumin, but also plasma proteins present at lower concentrations, such as fibrinogen. When enough liquid escapes from the vessels, liquid begins to enter the alveoli, a condition known as alveolar edema. In flooded alveoli, the air-liquid interface forms a meniscus. Thus, as described by the Laplace relation, flooded alveolar liquid pressure is less than alveolar air pressure, to a degree that is proportional to surface tension at the meniscal interface. The additional liquid in the airspace effectively thickens the alveolar-capillary barrier across which gas exchange occurs.

Further, with alveolar edema, there are regions of the lungs in which alveolar flooding is heterogeneous. That is, aerated and liquid-flooded alveoli are interspersed. ‘Intervening’ septa, i.e., those located between adjacent aerated and flooded alveoli, are thus subjected to a relatively high air pressure on one side and a relatively low liquid pressure on the other. The air-liquid pressure difference across the intervening septum, which equals the pressure difference across the meniscus of the flooded alveolus and is proportional to surface tension, causes the intervening septum to bow into the flooded alveolus. Thus, at any given lung inflation pressure, the intervening septum is extended beyond its normal length and becomes a site of stress concentration.

Patients with ARDS are treated by mechanical ventilation, which assists gas exchange but often causes an over-distension injury that exacerbates the underlying lung disease and prevents patient recovery. In particular, mechanical ventilation inflates the lung above the volumes reached during spontaneous breathing, thus increasing surface tension above normal and, as a result, exacerbates the surface tension-dependent stress concentrations in intervening septa between aerated and flooded alveoli. Consequently, mechanical ventilation of heterogeneously flooded regions injuriously increases permeability of an initially intact alveolar-capillary barrier to a degree that is proportional to the surface tension of the alveolar liquid in the region.

As over-distension injury is surface tension-dependent, lowering surface tension of the liquid in the alveoli of an edematous lung should directly lessen ventilation injury. Clinical surfactant therapy trials have tested intratracheal instillation of exogenous animal (either bovine, e.g., SURVANTA® (commercially available from Abbvie, Inc. located in North Chicago, Ill., U.S.A.), or porcine) surfactant as a means of lowering surface tension and treating ARDS. SURVANTA® is intended to provide surface-tension lowering properties similar to that of natural (endogenous) lung surfactant and is generally a mixture of bovine-harvested phospholipids, including DPPC, neutral lipids, fatty acids and surfactant proteins B and C, additional DPPC; palmitic acid; and tripalmitin, all suspended in a 0.9% sodium chloride solution. More specifically, SURVANTA® typically has a phospholipid concentration of about 25 milligrams per milliliter (mg/mL), a triglyceride concentration of from about 0.5 mg/mL to about 1.75 mg/mL, and a protein content of less than about 1.0 mg/mL. However, such exogenous surfactant therapy has not reduced ARDS mortality. One possible reason for the failure of exogenous surfactant therapy is heterogeneity of exogenous surfactant distribution throughout the lungs.

Further, the heterogeneous alveolar flooding pattern is attributable to liquid being trapped in discrete alveoli by a ‘pressure barrier,’ i.e., the presence of a higher liquid pressure at the edge than in the center of flooded alveoli. And the pressure barrier is no proportional to surface tension at the air-liquid interface. Lowering surface tension, by lowering the pressure barrier, can facilitate liquid escape from flooded alveoli and redistribution, in a more homogeneous fashion, across neighboring alveoli. Such liquid escape from flooded alveoli reduces alveolar flooding heterogeneity and should reduce the number of stress concentrations present. Thus lowering surface tension should also, by reducing flooding heterogeneity, indirectly reduce mechanical ventilation injury.

Cardiogenic pulmonary edema (CPE). In cardiogenic pulmonary edema, liquid entrance into the alveoli of the lung is driven not by abnormally elevated permeability of the alveolar-capillary barrier but, rather, by abnormally elevated pulmonary capillary blood pressure secondary to left heart dysfunction. As barrier no permeability is, at least initially, normal, plasma proteins should be trapped in the capillaries and plasma protein concentration in the alveolar edema liquid should be low. Yet, quantitative analysis of alveolar liquid in CPE demonstrates that protein concentration is elevated above normal in CPE, to the same degree as in ARDS. Further, in CPE, as in ARDS, there are regions of the lungs in which alveolar flooding is heterogeneous.

It may be that mechanical ventilation of CPE patients exacerbates stress concentrations in regions of heterogeneous alveolar flooding, thus injuring the alveolar-capillary barrier in such regions and leading to plasma protein entrance into the edema liquid. Regardless of the mechanism responsible for the elevated edema liquid plasma protein concentration in CPE patients, however, alveolar flooding pattern and edema liquid plasma protein concentration are similar between CPE and ARDS. In CPE, as in ARDS, lowering surface tension should, by either direct or indirect means, lessen ventilation injury of regions with heterogeneous alveolar flooding.

Neonatal respiratory distress syndrome (NRDS). Lung surfactant is produced during the third trimester of gestation and is critical to the ability of a baby to breathe unaided. Historically, many premature babies did not survive. Since the 1980's, tracheal instillation of exogenous animal surfactant has been a tremendously successful therapy that has enabled premature babies to live. However, there remains room for improvement in the clinical treatment of NRDS.

As the lungs are entirely filled with liquid prior to the first breath following birth, there are similarities between neonatal and edematous lungs. Neonatal respiratory distress is similar to CPE, in particular, in that both barrier permeability and alveolar liquid protein concentration are, initially, normal/low. However, with mechanical ventilation, barrier permeability and alveolar liquid protein content increase. This increase is likely attributable to mechanical ventilation causing heterogeneous aeration, thus leaving behind heterogeneous flooding and resulting in exacerbation of stress concentrations in heterogeneously flooded regions. An important difference between NRDS and both ARDS and CPE is that in NRDS there is less surfactant present than in mature lungs.

In NRDS, lowering surface tension with exogenous surfactant therapy is already beneficial. However lowering surface tension to a greater degree, or more uniformly throughout the lungs, should further lessen ventilation injury of regions with heterogeneous flooding.

Surface tension assessment methods. Surface tension is assessed in the isolated lung and in vitro using four complementary methods, as follows.

Method 1. Surface tension determination in the adult rat lung. In the isolated adult rat lung, a surface alveolus is micropunctured and a test solution, labeled with a low concentration of fluorescent dye verified not to alter surface tension, is instilled. In flooded alveoli, the air-liquid interface forms a meniscus, at which surface tension is determined as follows. Alveolar air pressure is determined with a transducer at the trachea of the constantly-inflated lung. Alveolar liquid phase pressure is determined by servo-nulling pressure measurement. Meniscus radius of curvature is determined by confocal microscopy. Surface tension is calculated according to the Lapalce relation.

Method 2. Ventilation ‘injury score’ in the adult rat lung. In the isolated, perfused adult rat lung, a surface alveolus is micropunctured and a non-fluorescent test solution is instilled. In experimental regions, a sufficiently large volume of liquid is instilled to generate a pattern of heterogeneous alveolar flooding; in control regions, a sufficiently small volume of liquid is instilled that the liquid spontaneously clears from the region, leaving behind a micropunctured-but-aerated region. Fluorescent dye, at a low concentration verified not to alter surface tension, is included in the perfusate. The region is imaged by confocal microscopy over a five minute baseline period at a constant transpulmonary pressure of 5 cm H₂O. Five ventilation cycles are supplied to the lung, at 0.33 Hz with a positive end-expiratory pressure of 15 cm H₂O and a tidal volume of 6 ml/kg body weight. The lung is then returned to a constant transpulmonary pressure of 5 cm H₂O and imaged for 10 additional minutes. Alveolar liquid fluorescence at all time points is normalized by capillary fluorescence.

At baseline, alveolar liquid fluorescence (in flooded alveoli of experimental regions or in the liquid lining layer of control, aerated regions) is low and constant in all regions. Following ventilation, alveolar liquid fluorescence remains unchanged in aerated regions but continually increases with time in heterogeneously flooded regions. This result indicates that in heterogeneously flooded, but not aerated, regions, ventilation injures the alveolar-capillary barrier, permitting fluorescence to pass from the vascular perfusate to the alveolar liquid, and the injury is sustained over time. The increase above baseline in normalized alveolar liquid fluorescence at the last time point of the experiment is used as an injury score. The injury score, which indicates the rate of increase of normalized fluorescence following ventilation, correlates with surface tension of the test solution.

Method 3. Opening pressure of the immature fetal rat lung. To inflate the initially liquid-filled fetal lung for the first time, the pressure applied at the trachea must be sufficient to overcome a capillary force that is proportional to surface tension of the liquid in the lung. Thus, following instillation of a test solution in the trachea of the immature fetal rat lung, the opening pressure of the lung is indicative of the surface tension of the test solution. At embryonic day 18 or 19 (term=day 22), a fetus is 185 delivered from a pregnant rat by uterotomy. (The normalized phospholipid content of the fetal rat lung on embryonic day 19 is 65% of that at full term.) A test solution (4-5 μl) is placed in the tip of a cannula; the cannula is inserted into the trachea and fixed in place with a suture; and a column of water, behind an air-filled cylinder that is connected to the tracheal cannula, is used to raise tracheal pressure in 10 cm H₂O steps. The opening pressure that causes air to flow into the lungs is recorded, and is proportional to test solution surface tension.

Method 4. Surface tension in a liquid drop. In a 3 μl drop of liquid (normal saline +31 μM fluorescein, which does not alter surface tension, for fluid visualization+test solutes), surface tension is determined using the same method as in the adult rat lung (method #1, above). Liquid pressure in the drop is determined by servo-nulling pressure measurement; interfacial radius of curvature is determined by confocal microscopy; and air pressure is known to be atmospheric. Surface tension is calculated according to the Laplace relation and found to be 72±2 mN/m for normal saline, as expected.

Surfactant therapy limitations. As noted above, surfactant therapy is successful in premature neonates but has not reduced mortality in ARDS. Even in neonates, there is room for improvement of surfactant therapy. As also noted above, the fact that high plasma protein concentrations are present in the alveolar liquid of premature neonates suggests that aeration, despite surfactant therapy, is sufficiently heterogeneous that stress concentrations are present and exacerbated by mechanical ventilation, resulting in injury to the alveolar-capillary barrier in NRDS. In translating surfactant therapy from neonates to adults while maintaining the same surfactant dosage per kg of body weight, the quantity of surfactant required becomes excessive. Use of a dilute surfactant would reduce the quantity of surfactant required. Further, there is evidence that dilute surfactant solutions distribute more homogeneously throughout the lungs than do concentrated solutions, which could be beneficial to both neonates and adults.

There are concerns about the use of animal surfactant, which include the possibility that it contains prions, which may cause brain disease. Thus attempts have been made to produce a synthetic surfactant. A synthetic surfactant could be the combination of lipids with one or more recombinant or synthetic surfactant protein. Efforts have focused on recombinant and synthetic forms of SP-C.

Surfactant protein C. SP-C is a 4.2 kilodalton (kD), 34 amino acid peptide. It has an α-helix and an N-terminal region of undefined conformation. Two 220 cysteine residues in the N-terminal region are palmitoylated. Various forms of recombinant SP-C and synthetic SP-C (sSP-C) have been identified and/or tested as a component of synthetic surfactant, in which the role of the recombinant or synthetic SP-C would be to promote lipid adsorption. One such form is the unpalmitoylated sSP-Css-ion lock (GIPSSPVHLKRLLIVVVVVELIVKVIVGALLMGL (SEQ ID NO. 1)) which is disclosed in U.S. Patent Application Publication No. 2015/0125515, which is hereby incorporated herein by reference. In this peptide, serine residues are substituted for the two cysteines, to avoid cross bridge formation and aggregation in the absence of palmitoylation. Additionally, a glutamine with a negatively charged side chain and a lysine with a positively charged side chain are substituted within the α-helix region at residues 20 and 24, respectively. The oppositely charged side chains, located approximately one turn of the a-helix apart, are thought to attract one another and thus form an ‘ion lock’ that stabilizes the α-helix. Alternatively, also as disclosed in U.S. Patent Application Publication No. 2015/0125515, phenylananine residues may be substituted for the two cysteins, to avoid cross bridge formation and aggregation in the absence of palmitoylation. And leucines may be substituted for valines in the a-helix region. Leucines, with longer side chains than valines, help maintain a-helix integrity.

Novel findings. Although, to date, no synthetic surfactant has functioned as well as animal surfactant, Applicants have surprisingly found that low concentrations of surfactant, isolated SP-C or isolated sSP-C, in the presence of albumin, can lower surface tension in the lungs and thereby minimize mechanical ventilation injury to an edematous lung. Applicants have tested 1% SURVANTA® solution in conjunction with albumin and alternative negatively charged solutes; human SP-C isolated from pulmonary alveolar proteinosis patients, with albumin; sSP-Css-ion lock, with albumin; sSP-Css-ion lock-B, a variant of sSP-Css-ion lock with a biotinylated N-terminal, with albumin; and sSP-Cff-leuc (GIPFFPVHLKRLKLLLLLLLLILLLILGALLMGL (SEQ ID NO. 2)), in which phenylananine residues are substituted for the two cysteins and leucines are substituted for valines in the a-helix region, with albumin. It is believed that besides albumin, an alternative negatively charged solute, such as fibrinogen or negatively charged 70 kD dextran, would cooperate with low concentrations of isolated SP-C or sSP-C to lower surface tension in the lungs and thereby minimize mechanical ventilation injury to an edematous lung. Based on this finding, it is believed that a recombinant or synthetic SP-C, alone, could constitute a synthetic surfactant that could achieve the aforesaid goal. It is noted that, at 4.2 kD, which is smaller than the 66 kD albumin that passes from capillary to alveolus in ARDS, CPE, and NRDS, isolated SP-C could be delivered intravascularly, potentially increasing either the homogeneity of the therapy throughout the lungs or the matching of the therapy to the edematous regions that require it. The sequence listings for unpalmitoylated sSP-Css-ion lock and sSP-Cff-leuc are presented in Table 1 hereinbelow.

SUMMARY OF THE INVENTION

Methods of the present invention comprise lowering the surface tension of liquid in alveoli of the edematous lung by providing to the alveoli a surfactant-associated protein and a negatively charged solute. Dilute surfactant protein C (SP-C) interacts with albumin or another negatively charged solute, such as fibrinogen or negatively charged 70kD dextrin, to lower surface tension. The negatively charged solute may be already present or may be added with the SP-C. SP-C could be administered via either the trachea or the vasculature. Lowering the surface tension of alveolar liquid will reduce ventilation injury of the heterogeneously flooded lung.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference is made to the following detailed description of an exemplary embodiment considered in conjunction with the accompanying drawings, in which:

FIG. 1A. Injury Score for Flooding Solutions in the Adult Rat Lung. In 275 control, aerated regions, instilled solutions are 0-5% albumin, 5% 70 kD dextran, 10 μM NaOH or 5% 70 kD dextran plus 10 μM NaOH, all in normal saline, without (n=24) or with (n=24) 1% SURVANTA®. These solutions have surface tensions spanning the full range tested. In flooded regions, instilled liquid is normal saline with additives as specified (n=4/group). When SURVANTA® is included, its concentration is 1%. Statistics: data shown as mean±standard deviation. All groups with flooding differ from control, aerated groups (p<0.01, statistics not shown on graph); *p<0.01 vs. no SURVANTA® for same flooding liquid; #p<0.01 vs. 3% albumin plus SURVANTA® and p<0.05 vs. 5% fibrinogen plus SURVANTA®.

FIG. 1 B. Surface Tension for Flooding Solutions in the Adult Rat Lung. Surface tension in alveoli flooded with normal saline plus 31 μM fluorescein and additives, as specified (n≥4/group). SURVANTA® concentration is 0.9%. Statistics: *p<0.05 vs. no SURVANTA® for same flooding liquid; #p<0.01 vs. no SURVANTA® for same flooding liquid.

FIGS. 1C and 1D. Injury Score and Surface Tension for Flooding Solutions with Albumin Concentrations at the High End of the Effective Range in the

Adult Rat Lung. Flooding solution is normal saline plus solutes as specified and, in surface tension-determination experiments, 31 μM fluorescein. SURVANTA® concentrations are 1% for ventilation injury experiments and 0.9% for surface tension determination experiments. Control groups combine data for two albumin solutions—in the absence of SURVANTA®—whose albumin concentrations bracket those of the solutions tested with SURVANTA® and between which there is no difference in injury score or surface tension. Statistics: *p<0.05 vs. control group without SURVANTA®; #p<0.01 vs. control group without SURVANTA®.

FIG. 1E. Injury Score Data Plotted vs. Surface Tension Data. Open symbol is average of data groups between which neither injury score nor surface tension differ. R²=0.65.

FIG. 2A. Injury Score for Flooding Solutions Containing Specific Surfactant Components in the Presence of Albumin. Solutes are at about the same concentrations as present in 1% SURVANTA® solution. The SP-B and SP-C used are isolated from pulmonary alveolar proteinosis patients; the SP-C, thus, is likely a mixture of fully-, partially- and non-palmitoylated peptide. Two forms of synthetic SP-C are used. One is sSP-Cff-leuc. The other is sSP-Css-ion lock-B. Only SP-C and sSP-C lower injury score, thus lower surface tension. Base solution for all groups is normal saline with 5% albumin. Due to pre-dissolution of certain solutes, at high concentration, in non-aqueous solvents, the final DPPC solution contains 2% methanol; the final SP-B and SP-C solutions contain 1.6% chloroform and 0.8% methanol; and the final sSP-Cff-leuc solution contains 1.3% chloroform and 1.3% ethanol. The sSP-Css-ion lock-B is dissolved directly in 5% albumin solution in normal saline, without additional solvents. n=1 or 2/group. From these results it appears that albumin facilitation of dilute SURVANTA® solution is attributable to albumin-SP-C interaction.

FIG. 2B. Injury Score for Flooding Solutions Containing SP-C, With and Without Albumin and DPPC. In normal saline without albumin, SP-C fom pulmonary alveolar proteinosis patients loses its ability to lower injury score, thus lower surface tension. Inclusion of DPPC does not restore the ability of SP-C to lower injury score, thus surface tension, in the absence of albumin. Base solution for all groups is normal saline. Due to pre-dissolution of certain solutes, at high concentration, in non-aqueous solvents, the final SP-C solutions, with or without albumin, contain 1.6% chloroform and 0.8% methanol; and the final SP-C plus DPPC solution contains 1.6% chloroform and 2.8% methanol. n =1 or 2/group. From these results it appears that isolated SP-C is surface active only when facilitated by albumin.

FIG. 3. Opening Pressure for Tracheal Instillation Solutions in the Immature Fetal Rat Lung. Solution (4-5 μl), with solutes as specified, is instilled in the trachea of the fluid-filled immature (embryonic day 18 or 19) fetal rat lung. The pressure required to inflate the fetal rat lung for the first time is proportional to surface tension. Base solution is normal saline excepting that base solution is Ringer's solution for 1% SURVANTA® plus 5% albumin. The solution of 0.0005% SP-C plus 5% albumin additionally includes 1.6% chloroform and 0.8% methanol. The sSP-Css-ion lock used is not biotinylated and is dissolved directly in normal saline without additional solvents. n=1 or 2/group.

FIG. 4. Surface Tension for Solutions In Vitro. Surface tension of normal saline drops (3 μl) containing 31 μM fluorescein and additional solutes as specified. The SP-C solution additionally contains 1.6% chloroform and 0.8% methanol. The fourth, sixth and seventh bars show that, in the presence of 5% albumin, surface tension decreases with increasing SURVANTA® concentration. The third, fourth and fifth bars demonstrate that there is an optimal albumin concentration of ˜5% for the facilitation of 1% SURVANTA®. n=2 or 3/group.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENT

The present invention overcomes the disadvantages and shortcomings discussed above. Four alternative methods are used to assess the surface activity of test solutions:

1. direct surface tension determination in surface alveoli of the isolated adult rat lung, which contains native lung surfactant, following alveolar instillation of a test solution;

2. degree of ventilation induced injury in a region of the isolated, perfused adult rat lung in which surface alveoli have been flooded, in a heterogeneous fashion, with a test solution;

3. initial inflation pressure of the immature fetal rat lung, which contains a reduced quantity of native surfactant compared with the adult lung, following tracheal instillation of a test solution; and 4. surface tension determination in a normal saline drop containing known quantities of additional solutes and no lipids other than any that are added to it.

The concentrations of SURVANTA discussed below are in volume percent (vol %) based on the total volume of the liquid in which the SURVANTA is dispersed. The concentrations of SP-C, whether natural, recombinant or synthetic, discussed below are in weight/volume percent (w/v %) based on the weight (in grams) of SP-C dispersed and the volume (in tenths-of-liters) of liquid in which the SP-C is dispersed.

The concentrations of the negatively charged solute (e.g., albumin, fibrinogen or negatively charged 70 kD dextrin) discussed below are also in weight/volume percent (w/v %) based on the weight (in grams) of the negatively charged solute dispersed and the volume (in tenths-of-liters) of liquid in which the negatively charged solute is dispersed.

In practice, with human patients having an edematous lung and receiving mechanical ventilation, the volume of liquid of concern in the body (i.e., the volume of liquid in which either SP-C or a negatively charged solute, or both, is to be dispersed) is understood to be the sum of the edema liquid and blood plasma. Persons of ordinary skill in the art will be familiar with this principle and capable of calculating an estimated volume for a particular patient in need of receiving treatment, as well as calculating the therapeutically effective amount of SP-C or negatively charged solute necessary which will provide the concentrations discussed below.

The following discussion is intended to provide guidance without limiting the methods described and contemplated herein. Functional residual capacity (FRC), which is the air volume in the lung at the end of expiration, averages 2.3 liters (L) in humans. In pulmonary edema, permeability of the lung capillaries is elevated such that solutes, such as SP-C and a negatively charged solute, can pass between the alveolar edema liquid and the blood plasma. Therefore, the plasma volume, which averages 3 L, should be included in the calculation of the volume of concern. As recognized by persons of ordinary skill in the art, a particular human patient's weight and hematocrit values will aid in estimating the plasma volume.

Assuming that, in a human patient with pulmonary edema, somewhere between 5 and 80% of FRC were flooded with liquid, then the total volume of edema liquid would be 0.12-1.8 L (based on the average 2.3 L mentioned above) and the total volume of edema liquid plus blood plasma would be 3.1-4.8 L. This would be the total volume of the liquid of concern upon which to base further calculations of the range of amounts of SP-C and the negatively charged solute that would be required to be therapeutically effective at providing the concentrations discussed below. More particularly, therapeutically effective amounts of SP-C and the negatively charged solute are those amounts that, directly or indirectly, minimize mechanical ventilation injury to an edematous lung by reducing the surface tension of alveolar liquid so that stress concentrations and alveolar flooding heterogeneity are reduced. As surprisingly discovered and described herein, therapeutically effective amounts of SP-C and the negatively charged solute are those amounts that provide the concentrations of these substances in the volume of liquid of concern as discussed hereinbelow because those concentrations of SP-C and the negatively charged solute reduce the surface tension of alveolar liquid.

From the results of testing based on the four methods described in the background above, it has been found that:

1. A dilute solution of 1 vol % SURVANTA® in normal saline is not surface 405 active, but that solutions of 1-5 vol % SURVANTA® are surface active when facilitated by 5% albumin solution. Further, based on the test results reported in FIG. 2, it is the SP-C in SURVANTA® that interacts with albumin; solutions of various forms of natural and synthetic SP-C at concentrations comparable to that in 1 vol % SURVANTA® are surface active in the adult or fetal rat lung, but only when facilitated by albumin solution. A solution of SP-C plus albumin tends to lose its surface activity in vitro in the absence of lipids, however, suggesting the SP-C and albumin, together, may reduce surface tension by promoting the adsorption of surfactant lipids.

Dilute surfactant solution or SP-C solution, where the SP-C may be natural, recombinant or synthetic, could be administered intratracheally in some embodiments. With sufficient albumin present in the alveolar liquid, dilute surfactant or SP-C solution could simply be administered in buffer (normal saline, Ringer's solution, physiologic saline solution, or equivalent). Without sufficient albumin present, a facilitating negatively charged solute (e.g., albumin, fibrinogen, negatively charged dextran or alternative negatively charged solute) could be added to the administered composition. The surfactant in the dilute surfactant solution may be SURVANTA® or another surfactant isolated from an animal that comprises SP-C. Suitable recombinant or synthetic SP-C are reasonably believed to include any of those identified in U.S. Patent Application Publication No. 2015/0125515, which has already been mentioned and incorporated herein by reference above.

Dilute surfactant solution or SP-C solution, where the SP-C may be natural, recombinant or synthetic, could alternatively be administered intravascularly, in the absence or presence of exogenous albumin or of an alternative negatively charged facilitating solute. Again, the surfactant in the dilute surfactant solution may be SURVANTA® or another surfactant isolated from an animal that comprises SP-C.

2. Albumin or alternative negatively charged solutes (e.g., fibrinogen, negatively charged 70 kD dextran) facilitate the surface activity of dilute SURVANTA® solution containing SP-C. The surface activity of the dilute SURVANTA® solution in normal saline was assessed by three of the four surface tension determination methods discussed in the background above.

Albumin concentrations of 3-11 w/v % facilitate the surface activity of 1 vol % SURVANTA® solution in the adult rat lung. Alternatively, 5 w/v % fibrinogen or 5 w/v % negatively charged 70 kD dextran (negative charge imparted by inclusion of 10 μM NaOH) also facilitate 1 vol % SURVANTA®. In contrast, 5 w/v % neutral 70 kD dextran does not facilitate 1 vol % SURVANTA®. Thus osmotic pressure is not sufficient to facilitate 1 vol % SURVANTA®; a negatively charged solute is required. Control experiments have shown 10 μM NaOH alone, without dextran, has no effect on surface tension or lung injury in the absence or presence of SURVANTA® (see FIGS. 1A and 1B).

In vitro, albumin is likewise required to facilitate the surface activity of SURVANTA®. However, the albumin concentration range that facilitates 1 vol % SURVANTA® does not extend to 10 vol %. Further, as shown by the data in FIG. 4, dose-response experiments demonstrate that, in conjunction with 5 w/v % albumin, 5 vol % SURVANTA® lowers surface tension more than 1 vol % SURVANTA®.

3. Surfactant protein C is the SURVANTA® component that interacts with 450 albumin to lower surface tension. SURVANTA® contains 2.5% total phospholipids, which includes 1.1-1.6% DPPC, and <0.1% of SP-B and SP-C combined, with a concentration of SP-C that is up to 15 times that of SP-B. Thus 1% SURVANTA® solution contains 0.025% total phospholipids, ˜0.01% DPPC, <0.001% SP-C and <<0.001% SP-B. The surface activity of solutions containing DPPC, SP-B and SP-C, in concentrations comparable to those in 1 vol % SURVANTA® solution, was assessed by ventilation injury assay (method #2). In conjunction with 5 w/v % albumin solution, only solutions with SP-C or sSP-C lower injury score, thus surface tension. Surfactant protein C alone, without albumin, is not surface active. Neither is the combination of SP-C and DPPC, in the absence of albumin, surface active.

4. The combination of SP-C and albumin lowers surface tension in the presence of at least a low concentration of surfactant lipids. The combination of SP-C, or sSP-C, and albumin lowers surface tension in the adult rat lung with normal levels of native surfactant and in the immature fetal rat lung with reduced surfactant levels. The combination of SP-C and albumin is likewise surface active in in vitro tests of dilute 1-5 vol % SURVANTA® containing only 0.03-0.13 w/v % total phospholipids (compared with 2.5 w/v % in undiluted SURVANTA®). Thus the combination of SP-C and albumin is surface active in the presence of low lipid concentrations. However, the combination of SP-C and albumin in the absence of any lipids demonstrates only low surface activity in vitro. The combination of SP-C and albumin appears to be highly effective at promoting 470 lipid adsorption, but to require the presence of at least a low lipid level for surface activity.

By extension of the above findings, it is expected that a concentration of from greater than about 2 w/v % to less than about 12 w/v % of a negatively charged solute (e.g., albumin, fibrinogen, and negatively charged 70 kD dextran), will facilitate the surface activity of a concentration of at least 0.01 vol % SURVANTA® or other surfactants isolated from animals; or of from about 0.000001 w/v % to about 1 w/v % SP-C, whether natural, recombinant or synthetic, in the presence of at least low levels of surfactant lipids, the relative proportions of which might be the same as or different from that in natural lung surfactant. This effect is reasonably expected regardless of whether the negatively charged solute is included in the delivered composition or already present, for example, in edema liquid or blood plasma. The recombinant or synthetic SP-C peptides that could be used include sSP-Css ion lock; sSP-Css ion lock-B; sSP-Css-ion lock with biotin tags(s) in alternative locations; sSP-Cff-leuc; biotinylated sSP-Cff-leuc; or alternative variations of natural human or animal SP-C.

In some embodiments, the therapeutically effective concentration of SP-C (natural, recombinant or synthetic) in the liquid of concern may be, for example without limitation, from about 0.00001 w/v % to about 1 w/v %, or from about 0.0005 w/v % to about 1 w/v %, or from about 0.0001 w/v % to about 1 w/v %, or from about 0.005 to about 1 w/v %, or from about 0.0025 w/v % to about 1 w/v %, or from about 0.00001 w/v % to about 0.05 w/v %, or from about 0.0005 w/v % to about 0.05 w/v %, or from about 0.0001 w/v % to about 0.05 w/v %, or from about 0.005 to about 0.05 w/v %, or from about 0.0025 w/v % to about 0.05 w/v %, 0.00001 w/v % to about 0.01 w/v %, or from about 0.0005 w/v % to about 0.01 w/v %, or from about 0.0001 w/v % to about 0.01 w/v %, or from about 0.005 to about 0.01 w/v %, or from about 0.0025 w/v % to about 0.01 495 w/v %, or from about 0.00001 w/v % to about 0.1 w/v %, or from about 0.0005 w/v % to about 0.1 w/v %, or from about 0.0001 w/v % to about 0.1 w/v %, or from about 0.005 to about 0.1 w/v %, or from about 0.0025 w/v % to about 0.1 w/v %,

In some embodiments, the therapeutically effective concentration of the negatively charged solute in the liquid of concern may be, for example without limitation, from about 2.1 w/v % to about 11.9 w/v %, or from about 2.5 w/v % to about 11.9 w/v %, or from about 3 w/v % to about 11.9 w/v %, or from about 4 or from about 3 w/v % to about 11.9 w/v %, or from about 5 w/v % to about 11.9 w/v %, or from about 6 w/v % to about 11.9 w/v %, or from about 2.1 w/v % to about 11.5 w/v %, or from about 2.1 w/v % to about 11 w/v %, or from about 2.1 w/v % to about 10 w/v %, or from about 2.1 w/v % to about 9 w/v %, or from about 2.5 w/v % to about 11.5 w/v %.

TABLE 1 SEQUENCE LISTING <110> Stevens Inst. of Tech. Perlman, Carrie <120> DILUTE SURFACTANT OR ISOLATED SURFACTANT PROTEIN SOLUTION FOR THE REDUCTION OF SURFACE TENSION IN THE LUNG <150> US 15/194,096 <151> 2016 Jun. 27 <130> 101995.043102 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> sSP-Css-ion lock <400> 1 Gly Ile Pro Ser Ser Pro Val His Leu Lys Arg Leu 1               5                   10 Leu Ile Val Val Val Val Val Glu Leu Ile Val Lys         15                  20           Val Ile Val Gly Ala Leu Leu Met Gly Leu  25                 30  <210> 2 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> sSP-Cff-leuc <400> 2 Gly Ile Pro Phe Phe Pro Val His Leu Lys Arg Leu 1               5                   10  Lys Leu Leu Leu Leu Leu Leu Leu Leu Ile Leu Leu         15                  20  Leu Ile Leu Gly Ala Leu Leu Met Gly Leu 25                  30      

1. A method of reducing ventilation injury to a patient whose lung has regions with heterogeneous alveolar flooding by alveolar liquid, said method comprising the step of (i) delivering to the patient a solution comprising a predetermined amount of a surfactant protein C, wherein said predetermined amount of said surfactant protein C is selected to provide a concentration of said surfactant protein C in the alveolar liquid in a range of from 0.000001 weight/volume percent to 1 weight/volume percent after the performance of step (i), and wherein a negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from greater than 2 weight/volume percent to less than 12 weight/volume percent after the performance of step (i), said weight/volume percents of said surfactant protein C and said negatively-charged solute being based on the total volume of the alveolar liquid after the performance of step (i).
 2. The method of claim 1, wherein said negatively-charged solute comprises at least one chemical selected from the group consisting of albumin, fibrinogen and negatively-charged dextran.
 3. The method of claim 1, wherein said surfactant protein C is natural, recombinant, or synthetic.
 4. The method of claim 3, wherein said surfactant protein C is synthetic and selected from sSP-Css-ion lock, sSP-Css-ion sSP-Cff-leuc, and combinations thereof.
 5. The method of claim 1, wherein said concentration of said surfactant protein Cin the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 1 weight/volume percent.
 6. The method of claim 1, wherein said concentration of said surfactant protein C the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 1 weight/volume percent.
 7. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.1 weightivolunie percent.
 8. The method of claim 1, wherein said negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from 2.1 weight/volume percent to 11 weight/volume percent after the performance of step (i).
 9. The method of claim 1, wherein said surfactant protein C is derived from an animal.
 10. The method of claim 1 wherein said solution comprises a surfactant containing said surfactant protein C.
 11. The method of claim 1, wherein said solution further comprises lipids.
 12. The method of claim 1, wherein said alveolar liquid is contained in a patient's alveoli, said alveolar liquid having a surface tension which is lowered by the performance of step (i) such that the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of the patient's lung.
 13. The method of claim 12, wherein the lowered surface tension of the alveolar liquid reduces stress concentrations in the patient's lung.
 14. The method of claim 13, wherein the patient's alveoli are flooded to substantially the same extent.
 15. The method of claim 13, wherein the patient's alveoli are heterogeneously flooded, whereby the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of intervening septa located between aerated and flooded alveoli.
 16. The method of claim 1, wherein step (i) comprises the step of administering said solution comprising said surfactant protein C to a trachea or bronchus of the patient having the lung.
 17. The method of claim 1, wherein some of said negatively-charged solute is already present in the alveolar liquid and some of said negatively-charged solute is delivered as part of said solution.
 18. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume % to 0.1 weight/volume percent.
 19. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.05 weight/volume percent.
 20. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.05 weight/volume percent.
 21. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.05 weight/volume percent.
 22. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.05 weight/volume percent.
 23. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.05 weight/volume percent.
 24. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.01 weight/volume percent.
 25. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.01 weight/volume percent.
 26. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.01 weight/volume percent.
 27. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.01 weight/volume percent.
 28. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.01 weight/volume percent.
 29. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.1 weight/volume percent.
 30. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.1 weight/volume percent.
 31. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.1 weight/volume percent.
 32. The method of claim 1, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.1 weight/volume percent.
 33. The method of claim 1, wherein step (i) is performed by injecting said solution into a circulatory system of a patient having the lung.
 34. A method of reducing ventilation injury to a patient whose lung has regions with heterogeneous alveolar flooding by alveolar liquid which contains a negatively-charged solute, said method comprising the step of (i) delivering to the patient a solution comprising a predetermined amount of a surfactant protein C and no additional negatively-charged solute, wherein said predetermined amount of said surfactant protein C is selected to provide a concentration of said surfactant protein C in the alveolar liquid in a range of from 0.000001 weight/volume percent to 1 weight/volume percent after the performance of step (i), and wherein the negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from greater than 2 weight/volume percent to less than 12 weight/volume percent after the performance of step (i), said weight/volume percents of said surfactant protein C and said negatively-charged solute being based on the total volume of the alveolar liquid after the performance of step (i).
 35. The method of claim 34, wherein said negatively-charged solute comprises at least one chemical selected from the group consisting of albumin, fibrinogen and negatively-charged dextran.
 36. The method of claim 34, wherein said surfactant protein C is natural, recombinant, or synthetic.
 37. The method of claim 36, wherein said surfactant protein C is synthetic and selected from sSP-Css-ion lock, sSP-Css-ion lock B sSP-Cff-leuc, and combinations thereof.
 38. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 1 weight/volume percent.
 39. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 1 weight/volume percent.
 40. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.1 weight/volume percent.
 41. The method of claim 34, wherein said negatively-charged solute is present in the alveolar liquid in a concentration which is in a range of from 2.1 weight/volume percent to 11 weight/volume percent after the performance of step (i).
 42. The method of claim 34, wherein said surfactant protein C is derived from an animal.
 43. The method of claim 34, wherein said solution comprises a surfactant containing said surfactant protein C.
 44. The method of claim 34, wherein said solution further comprises lipids.
 45. The method of claim 34, wherein said alveolar liquid is contained in a patient's alveoli, said alveolar liquid having a surface tension which is lowered by the performance of step (i) such that the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of the patient's lung.
 46. The method of claim 45, wherein the lowered surface tension of the alveolar liquid reduces stress concentrations in the patient's lung.
 47. The method of claim 46, wherein the patient's alveoli are flooded to substantially the same extent.
 48. The method of claim 46, wherein the patient's alveoli are heterogeneously flooded, whereby the lowered surface tension of the alveolar liquid lessens ventilation-induced overdistension injury of intervening septa located between aerated and flooded alveoli
 49. The method of claim 34, wherein step (i) comprises the step of administering said solution comprising said surfactant protein C to a trachea or bronchus of the patient having the king.
 50. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume % to 0.1 weight/volume percent.
 51. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.05 weight/volume percent.
 52. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.05 weight/volume percent.
 53. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.05 weight/volume percent.
 54. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.05 weight/volume percent.
 55. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.05 weight/volume percent.
 56. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.00001 weight/volume percent to 0.01 weight/volume percent.
 57. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.01 weight/volume percent.
 58. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.01 weight/volume percent.
 59. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.01 weight/volume percent.
 60. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.01 weight/volume percent.
 61. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0005 weight/volume percent to 0.1 weight/volume percent.
 62. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0001 weight/volume percent to 0.1 weight/volume percent.
 63. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.005 weight/volume percent to 0.1 weight/volume percent.
 64. The method of claim 34, wherein said concentration of said surfactant protein C in the alveolar liquid after the performance of step (i) is in a range of from 0.0025 weight/volume percent to 0.1 weight/volume percent.
 65. The method of claim 34, wherein step (i) is performed by injecting said solution into a circulatory system of a patient having the lung. 